Bacterial cells, such as E. coli, are commonly used for producing recombinant proteins. There are many advantages to using bacterial cells, such as E. coli, for producing recombinant proteins particularly due to the versatile nature of bacterial cells as host cells allowing the gene insertion via plasmids. E. coli have been used to produce many recombinant proteins including human insulin.
Despite the many advantages to using bacterial cells to produce recombinant proteins, there are still significant limitations including the difficulty of producing protease sensitive proteins. Proteases play an important role in turning over old and miss-folded proteins in the E. coli periplasm and cytoplasm. Bacterial proteases act to degrade the recombinant protein of interest, thereby often significantly reducing the yield of active protein.
A number of bacterial proteases have been identified. In E. coli proteases including Protease III (ptr), DegP, OmpT, Tsp, prlC, ptrA, ptrB, pepA-T, tsh, espc, eatA, clpP and lon have been identified.
The Protease III (ptr) protein is a 110 kDa periplasmic protease which degrades high molecular weight proteins.
Tsp (also known as Prc) is a 60 kDa periplasmic protease. The first known substrate of Tsp was Penicillin-binding protein-3 (PBP3) (Determination of the cleavage site involved in C-terminal processing of penicillin-binding protein 3 of Escherichia coli; Nagasawa H, Sakagami Y, Suzuki A, Suzuki H, Hara H, Hirota Y. J Bacteriol. 1989 November; 171(11):5890-3 and Cloning, mapping and characterization of the Escherichia coli Tsp gene which is involved in C-terminal processing of penicillin-binding protein 3; Hara H, Yamamoto Y, Higashitani A, Suzuki H, Nishimura Y. J Bacteriol. 1991 August; 173 (15):4799-813) but it was later discovered that the Tsp was also able to cleave phage tail proteins and, therefore, it was renamed as Tail Specific Protease (Tsp) (Silber et al., Proc. Natl. Acad. Sci. USA, 89: 295-299 (1992)). Silber et al. (Deletion of the prc(tsp) gene provides evidence for additional tail-specific proteolytic activity in Escherichia coli K-12; Silber, K. R., Sauer, R. T.; Mol Gen Genet 1994 242:237-240) describes a prc deletion strain (KS1000) wherein the mutation was created by replacing a segment of the prc gene with a fragment comprising a Kanr marker.
DegP (also known as HtrA) is a 46 kDa protein having dual function as a chaperone and a protease (Families of serine peptidases; Rawlings N D, Barrett A J. Methods Enzymol. 1994; 244:19-61).
It is known to knockout bacterial proteases in order to affect the yield of recombinant protein.
Georgiou et al. (Construction and characterization of Escherichia coli strains deficient in multiple secreted proteases: protease III degrades high-molecular-weight substrates in vivo. Baneyx F, Georgiou G. J Bacteriol. 1991 April; 173(8):2696-703) studied the effects on growth properties and protein stability of E. coli strains deficient in protease III constructed by insertional inactivation of the ptr gene and observed an increase in the expression of a protease-sensitive secreted polypeptide. A strain comprising the ptr mutation and also deficient in the secreted protease DegP was also produced and found to have a decreased growth rate and an increase in protein expression. In Georgiou et al., the E. coli strains deficient in protease III and/or DegP were constructed from the KS272 parental strain which already comprises a number of genomic mutations.
U.S. Pat. No. 5,264,365 (Georgiou et al.) discloses the construction of protease-deficient Escherichia coli hosts which when combined with an expression system are useful for the production of proteolytically sensitive polypeptides.
Meerman et al. (Construction and characterization of Escherichia coli strains deficient in All Known Loci Affecting the Proteolytic Stability of Secreted Recombinant Proteins. Meerman H. J., Georgeou G., Nature Biotechnology, 1994 November; 12; 1107-1110) disclose E. coli strains comprising mutations in the rpoH, the RNA polymerase sigma factor responsible for heat shock protein synthesis, and different combinations of mutations in protease genes including DegP, Protease III, Tsp(Prc) and OmpT, where null mutations of the protease genes were caused by insertional mutations. In Meerman et al., the E. coli strains deficient in one or more of Tsp, protease III and DegP were constructed from the KS272 parental strain which already comprises a number of genomic mutations.
U.S. Pat. No. 5,508,192 (Georgiou et al.) discloses a method of producing recombinant polypeptides in protease-deficient bacterial hosts and constructs of single, double, triple and quadruple protease deficient bacteria which also carry a mutation in the rpoH gene.
Chen et al describes the construction of E. coli strains carrying different combinations of mutations in prc (Tsp) and DegP created by amplifying the upstream and downstream regions of the gene and ligating these together on a vector comprising selection markers and a sprW148R mutation (High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (ΔDegP Δprc spr W148R) host strain. Chen C, Snedecor B, Nishihara J C, Joly J C, McFarland N, Andersen D C, Battersby J E, Champion K M. Biotechnol Bioeng. 2004 Mar. 5; 85(5):463-74). The combination of the ΔDegP, Δprc and W148Rspr mutations were found to provide the highest levels of antibody light chain, antibody heavy chain and F(ab′)2-LZ. EP1341899 discloses an E. coli strain that is deficient in chromosomal DegP and prc encoding proteases DegP and Prc, respectively, and harbors a mutant spr gene that encodes a protein that suppresses growth phenotypes exhibited by strains harboring prc mutants.
Kandilogiannaki et al (Expression of a recombinant human anti-MUC 1 scFv fragment in protease-deficient Escherichia coli mutants. Kandilogiannaki M, Koutsoudakis G, Zafiropoulos A, Krambovitis E. Int J Mol Med. 2001 June; 7(6):659-64) describes the utilization of a protease deficient strain for the expression of a scFv protein. The protease deficient bacterial strains used previously to express recombinant proteins comprise further mutations of genes involved in cell metabolism and DNA replication such as, for example phoA, JhuA, lac, rec, gal, ara, arg, thi and pro in E. coli strains. These mutations may have many deleterious effects on the host cell including effects on cell growth, stability, recombinant protein expression yield and toxicity. Strains having one or more of these genomic mutations, particularly strains having a high number of these mutations, may exhibit a loss of fitness which reduces bacterial growth rate to a level which is not suitable for industrial protein production. Further, any of the above genomic mutations may affect other genes in cis and/or in trans in unpredictable harmful ways thereby altering the strain's phenotype, fitness and protein profile. Further, the use of heavily mutated cells is not generally suitable for producing recombinant proteins for commercial use, particularly therapeutics, because such strains generally have defective metabolic pathways and hence may grow poorly or not at all in minimal or chemically defined media.
Protease deficient bacterial strains also typically comprise knockout mutations to one or more protease encoding genes which have been created by insertion of a DNA sequence into the gene coding sequence. The inserted DNA sequence typically codes for a selection marker such as an antibiotic resistance gene. Whilst this mutation method may be effective at knocking out the target protease, there are many disadvantages associated with this method. One disadvantage is the insertion of the foreign DNA, such as an antibiotic resistance gene, causes disruption in the host's genome which may result in any number of unwanted effects including the over-expression of detrimental proteins and/or down-regulation or knockout of other essential proteins. This effect is particularly evident for those genes positioned immediately upstream or downstream of the target protease gene. A further disadvantage to the insertion of foreign DNA, particularly antibiotic resistance genes, is the unknown phenotypic modifications to the host cell which may affect expression of the target protein and/or growth of the host cell and may also make the host cell unsuitable for production of proteins intended for use as therapeutics. Antibiotic resistance proteins are particularly disadvantageous for biosafety requirements large scale manufacturing particularly for the production of therapeutics for human administration. A further disadvantage to the insertion of antibiotic resistance markers is the metabolic burden on the cell created by the expression of the protein encoded by the antibiotic resistance gene. The use of antibiotic resistance markers for use as markers for genetic manipulations such as knockout mutations, are also limited by the number of different antibiotic resistance markers available. Accordingly, there is still a need to provide new bacterial strains which provide advantageous means for producing recombinant proteins.